Role of omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes on intestinal barrier integrity and immunity in animals

The gastrointestinal tract of livestock and poultry is prone to challenge by feedborne antigens, pathogens, and other stress factors in the farm environment. Excessive physiological inflammation and oxidative stress that arises firstly disrupts the intestinal epithelial barrier followed by other components of the gastrointestinal tract. In the present review, the interrelationship between intestinal barrier inflammation and oxidative stress that contributes to the pathogenesis of inflammatory bowel disease was described. Further, the role of naturally existing immunomodulatory nutrients such as the omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes in preventing intestinal barrier inflammation was discussed. Based on the existing evidence, the possible molecular mechanism of these bioactive nutrients in the intestinal barrier was outlined for application in animal diets.


Introduction
Apart from nutrient absorption, the intestinal epithelial layer (IEL) acts as the first line of host defense against various environmental stress factors. Excessive physiological inflammation and oxidative stress primarily disrupts the inner lining of the gastrointestinal tract such as the IEL. Impairment of IEL can further contribute to the progression of inflammatory bowel diseases (IBD), metabolic disorders, and even mortality in animals [1][2][3]. Traditionally antibiotics are administered in animal diets to enhance the growth performance and prevent undesired inflammatory responses. Although antibiotics are beneficial to a certain extent, the development of antibiotic-resistance in bacterial populations is a major drawback [4,5]. Hence, there is a necessity for alternative, environmental-friendly immunomodulatory compounds to nurture animal health with limited side effects. Of note, certain bioactive compounds derived from plants, animals, and microbial sources exhibit therapeutic benefits beyond basic nutrition. The addition of these compounds to a certain level in an animal diet is reported to boost the gut immunity, confer stress resistance, and improve the overall health status [3,6,7]. The examples of such potential bioactive compounds include the traditional and emerging omega-3 polyunsaturated fatty acids (ω-3 PUFAs), citrus pectin (CPn), and the milk-derived exosomes (MDEs). In the previous in vitro and in vivo trials, these natural nutrients exhibited strong immunomodulatory, antioxidative, and antimicrobial properties in controlling chronic gut inflammation. This review describes the impact of inflammation and oxidative stress in the intestinal barrier that contributes to IBD. Further, the role of immunomodulatory nutrients as the ω-3 PUFAs, CPn and MDEs in controlling gut inflammation at the level of intestinal barrier is discussed for utilization in animal nutrition.

Intestinal epitheliuma dynamic barrier
Gastrointestinal tract is secured by a series of protective layers collectively described as the intestinal mucosa. It mainly encompasses the mucus layer, gut microbiota, IEL, immune cells dispersed in lamina propria and lamina. The IEL physically separates the circulatory system from the external milieu [ Fig. 1]. Besides, it is the key component that orchestrates gut homeostasis by establishing communication between the microbiota and the underlying immune cells [8,9]. Further, the IEL is formed by a monolayer of intestinal epithelial cells (IECs) interconnected by different protein complexes such as tight junction proteins, junction adherent proteins (JAM) and, desmosomes. The tight junction proteins such as occludens (OCLN) and claudins (CLDN) are the important structural units that strictly governs the permeability of molecules across the barrier [10]. Besides, the IEL comprises several specialized absorptive and secretory cell types for digestion, nutrition uptake, and host defense. Particularly, the enterocytes are the most abundant (~80%) and fast regenerating absorptive cell type [11,12]. Goblet cells secrete a gel-like glycoproteins called mucin (MUC) that forms a mucous layer above the IEL. Further, the mucus layer is embedded Fig. 1 Structure of intestinal epithelium. The intestinal epithelial layer (IEL) ① is the first lining of gastrointestinal tract. It is formed by a single layer of specialized intestinal epithelial cells (enterocytes, goblet cells, paneth cells, microfold cells, stem cells and enteroendocrine cells) that physically separates the gut lumen ② from the circulatory system. The IEL is lined by a mucous layer ③, where the gut microbiota ④ is embedded. The IEL orchestrates gut homeostasis by establishing communication between the gut microbiota and the underlying immune cells in lamina propria ⑤. The intestinal epithelial cells secrete several antimicrobial peptides ⑥ for host-defense. For references, see text. Figure created using BioRender.com with trillions of commensal bacteria called the gut microbiota. Paneth cells exhibit the longest life span and produce antimicrobial proteins such as defensins, lysozymes, and phospholipases [10,13]. The microfold cells regulate the adaptive immune responses by presenting the luminal pathogens and antigens to the immune cells of lamina propria [10]. Enteroendocrine cells primarily secrete hormones and other antimicrobial proteins for different physiological functions. The multipotent intestinal stem cells at the crypt base continuously regenerate to form other specialized cell types [11]. Altogether, the IEL forms a dynamic barrier that selectively permits the nutrients while block the detrimental factors as pathogens and toxins from entering into the circulatory system.

Immunomodulatory nutrients for intestinal health
The pursuance of reducing the antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs) has raised the interest in utilizing naturally existing bioactive compounds. These bioactive compounds are constituents of functional food, similar to conventional foods but provide additional therapeutic benefits. Thus, functional foods are described as the food prepared using 'scientific intelligence' vital for optimal health [6]. The European Food Safety Authority (EFSA) has categorized these natural bioactive or immunomodulatory compounds into, 'plant extracts', 'prebiotics', 'probiotics', 'animal-by products', and 'other substances' [22]. Plants have been the major source of energy since time immemorial in humans and animals. Certain compounds from whole or part of the plant exhibit immunomodulatory properties and are commonly referred to as 'phytobiotics' or 'botanicals' [23]. A classic example of a plant-or marinederived immunomodulatory compound is the essential fatty acid such as the ω-3 PUFAs. The common sources of ω-3 PUFAs are marine phytoplankton, linseed oil, and fish oil [24,25]. Since several decades, ω-3 PUFAs are routinely incorporated into human and animal diets in moderation for normal physiological functions. However, supplementation at a certain level result in achieving therapeutic effects. The ω-3 PUFA-enriched diet in livestock and poultry was shown to modulate the immune system, improve intestinal morphology, fertility, stress resistance, and performance [26][27][28][29].
The pectins from citrus fruit peels are increasingly recognized for its multiple health-beneficial properties. Although the application of CPn as a feed additive is emerging, preliminary studies have reported to exhibit potential anti-inflammatory, antimicrobial and prebiotic properties [30][31][32][33]. Besides, the food processing industries utilize CPn as gelling agent and stabilizers for ages and is regarded safe for consumption [34]. Moreover, GCS-100 and PectaSol-C are the two commercially available CPn-based drugs used for treating fibrosis in humans [35]. In the European Union, Italy is one of the leading producers of citrus fruits and after juicing, the peels are discarded as waste. These environmental wastes can otherwise be utilized as value-added, sustainable dietary supplements for animals. Another prominent, animal-based nutritive compound is the milkderived bioactive peptides and proteins [36]. Recently, the exosomes present in milk was identified to carry cargoes of nucleic acids and peptides that improves the immune system, promotes intestinal barrier development, and prevent inflammatory diseases [37][38][39][40]. Currently, the milk-derived exosomes (MDEs) are gaining attention for targeted inflammatory diseases through diet [40]. Unlike antibiotics, these natural bioactive compounds are effective, environmentally friendly, and safe for improvising the immune system of animals, while eliminating stress and discomfort. The immunomodulatory properties of ω-3 PUFAs, CPn, and MDEs at the intestinal-level, based on previous evidence are described in the following sections.

Omega-3 polyunsaturated fatty acids Structure and molecular mechanism
Although ω-3 PUFAs are widely known feed additive, its mechanism in intestinal barrier is poorly established, which limits the assessment of its efficacy. Based on previous studies, its possible modes of action are described presently. Generally, fatty acids are carbon chain structures of varying length, synthesized in the cytoplasm from Acetyl-CoA [41]. The two major fatty acid families as the omega-3 (ω-3) alpha-linolenic acid (ALA; C18:3n-3) and the omega-6 (ω-6) linoleic acid (LA; C18:2n-6) that cannot be synthesized by animals, but obtained from the diet are called essential fatty acids [24,42].

Impact on intestinal barrier under normal and stress conditions
In vitro studies The impact of ω-3 PUFAs on the intestinal barrier under normal and inflammatory conditions were previously evaluated using different IEC models (Table 1). Particularly, the enterocyte models were widely utilized as they are primarily involved in nutrition absorption, host-defence and are the most abundant cell type in IEL. In these experimental models, the pathophysiological events of barrier inflammation and oxidative stress was stimulated using different biological and chemical stressors. Some commonly applied stressors are pathogens, endotoxins, chemicals such as acetic acid, hydrogen peroxide (H 2 O 2 ), dextran sulfate sodium (DSS), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) [66,86]. Moreover, these models were widely reported to mimic the cardinal signs of IBD [86]. Accordingly, ω-3 PUFA (ALA or EPA) treatment modulated the integrity and permeability of human Caco-2 cell monolayer in a dose-dependent manner [56,57]. The epithelial integrity was determined by measuring the transepithelial electrical resistance (TEER) across the monolayer of IECs. Whereas, the barrier permeability was assessed by measuring the paracellular flux of small molecules such as horseradish peroxidase (HRP), fluorescein-5-(6)-sulfonic acid (FS), or fluorescein isothiocyanate-labelled dextran 4 kDa (FD4) between the tight junction gaps in the cell monolayer [56][57][58]. Further, pre-incubation of human T84 cells with ω-3 PUFA (ALA, EPA or DHA) secured the monolayer integrity and permeability from the damage against IL-4 pro-inflammatory cytokine as indicated by increased TEER values and decreased FD4 permeability compared to the control [58]. Similarly, the negative impact of heat-injury in the human Caco-2 cell monolayer was reduced by ω-3 PUFA (EPA or DHA) pre-treatment. Specifically, EPA highly enhanced the barrier integrity (TEER) and expressions of different tight junction proteins (Zonula occludens [ZO]-1, OCLN, CLDN-2), while decreased the barrier permeability to HRP flux compared to DHA [59].
For the first time, DHA-derived MaR1 and EPAderived RvE1 were shown to suppress the disease activity in murine colitis. Accordingly, MaR1 supplementation in DSS-colitis rats increased the expressions of different tight junction proteins (ZO-1, OCLN), while inhibited the mucosal damage, colon shortening, inflammatory cell infiltration, and expressions of different pro-   [72]. Importantly, MaR1 inhibited the protein expression of TLR4/NF-κB signaling (TLR4, p-NF-κB-p65) and instead activated the anti-inflammatory nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling [72]. Similarly, MaR1 intervention ameliorated the severity of acute and chronic DSS-colitis in mice by reducing epithelial damage, inflammatory cell infiltration, hyperaemia, colon wall thickness, colon shortening, and expression of pro-inflammatory genes or proteins (MPO, NF-κB, IL-1β, IL-6, TNF-α, interferon[INF]γ, ICAM-1) [75]. Likewise, RvE1 facilitated tissue repair in peritonitis mice with TNBS-colitis, while significantly reduced the leukocyte infiltration, MPO activity, and the expression of genes corresponding to pro-inflammatory response (COX-2, TNF-α, IL-12p40, iNOS) [76]. Generally, a steep oxygen gradient exists in the intestinal barrier to support the sustenance of gut microbiota and other barrier functions. An imbalance in the gradient can instigate barrier degenerative diseases such as ischemic-reperfusion and NEC [103]. Earlier, ω-3 PUFA remediated the experimental NEC induced by hypoxia, formula feeding, or its combination in murine models. Accordingly, DHA intervention significantly decreased tissue necrosis, incidence of disease, and the gene expression of pro-inflammatory mediators such as TLR2/4 and platelet-activating factor receptor (PAFR) in the NEC-rat pups [73]. Moreover, ω-3 PUFA (EPA or The arrow indicates an increase (↑) or decrease (↓) in the level or activity of the different parameters analysed, "=" symbol designates unchanged parameters. DHA) administration in maternal rats during gestation, significantly accumulated in the intestinal phospholipids of prematurely delivered rat pups. This subsequently ameliorated the impact of experimental NEC induced in rat pups by inhibited the gene expression of proinflammatory IκBα/β and instead activated the antiinflammatory PPARγ gene. Besides, the gene expression of PGE2 receptor EP3 and PGD2 receptor DP2 was increased, while the mucosal damage, inflammatory cell infiltration, and incidence of disease was decreased [74].
Apart from infections, weaning imparts tremendous inflammatory and metabolic stress in pigs due to changes in gastrointestinal physiology [104]. Previously, administration of fish oil in pigs significantly downregulated the protein expression of pro-inflammatory eicosanoids (PGE, PGI2, TXB2) through ω-3 PUFA enrichment in the intestinal phospholipids. Although the morphology of colonic mucosa remained stable, the crypt depth was significantly reduced [80] (Table 1). Further, fish oil administration during the course of gestation to lactation in sows markedly increased the ω-3 PUFA levels in ileal phospholipids. The maternal diet subsequently instigated an age-dependent decrease in villus height (d 0), crypt depth (d 7), and HRP permeability (d 0-28) in the piglet ileum. These effects were reported to prevent the intestinal pathologies associated with IEL functioning during the neonatal period [81].
In similar maternal physiology, supplementation of extruded linseed oil temporarily increased the FD4/LPS permeability in the piglet jejunum explants until d 28 and thereafter decreased at d 52 [82]. The distinct outcome of these two trials could have possibly aroused from a difference in linseed oil formulation administered, dose-effect, or animal physiology. Nevertheless, the influence of maternal dietary ω-3 PUFA on piglet intestinal development and health status is unclear and hence needs further investigations. In another study, fish oil administration in weaned piglets protected from LPS-induced damage on the intestinal barrier by enhancing the expressions of tight junction proteins (OCLN, CLDN-1), villus height, and V/ C ratio [83]. Besides, the expression of genes corresponding to the TLR4 pathway such as the TLR4, myeloid differentiation primary response 88 (MyD88), interleukin-1 receptor-associated kinase-1 (IRAK1), and TNFRassociated factor-6 (TRAF6) were suppressed. The expression of inflammatory genes as NOD2 and RIPK-2, and the expression of proteins as diamine oxidase, caspase-3, and heat shock protein-70 (HSP70) were also significantly downregulated [83]. Furthermore, fish oil supported the remission of DSS-colitis in the early-weaned piglets by enhancing enterocyte mitosis and gene expression of PPARγ-responsive uncoupling protein-3 (UPC3) [84]. Moreover, EPA intervention in piglets moderately supported the recovery of ileum ischemic injury by decreasing the expression of PGE2 protein and barrier flux to H 3mannitol/C 14 -inulin, while increased the expression of COX-2 gene and TEER values [85]. In another study that administered fish oil in pigs, exhibited a significant reduction in LPS permeability across the ileal explants [67].
In clinical settings, ω-3 PUFAs are administered either individually or as an immunological adjuvant for controlling different IBDs including Crohn's disease (CD), ulcerative colitis, and distal proctocolitis ( Table 2). Accordingly, in subjects with ethanol-induced duodenal injury, fish oil administration relieved macroscopic lesions by increased the protein expression of anti-inflammatory eicosanoid, LTC5 [87]. Also, EPA suppressed the mucosal expression of pro-inflammatory eicosanoid as LTB4 in pediatric ulcerative colitis under remission [88]. Further, administration of fish oil in patients with active ulcerative colitis or distal proctocolitis, witnessed a marked reduction in disease activity as observed from improvements in clinical, endoscopic, and histological scores [89][90][91][92]. In active ulcerative colitis or CD patients undertaking IBD medications, ω-3 PUFA (fish, fish oil or EPA) administration reduced the disease activity [93,95,96] and protein expression of pro-inflammatory eicosanoids (PGE2, PGI2, TXB2) [94] by incorporating in the mucosal phospholipids [93][94][95]. Similarly, the health status of ulcerative colitis patients in remission with or without IBD medication was improved by fish oil or EPA administration [97][98][99]. Specifically, the disease activity and mucosal inflammation were remediated by activating the gene expression of anti-inflammatory mediators such as the IL-10, and suppressor of cytokine signaling-3 (SOCS3), while reduced the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3). Additionally, the abundance of goblet cells, IL-22, and proteins that modulate the secretory and absorptive cell lineage such as the hairy and enhancer of split-1 (HES-1) and kruppel-like factor-4 (KLF-4) were also increased [99]. On the contrary, few studies have reported that ω-3 PUFA (fish oil, EPA, or DHA) had no beneficial effects in the patients with either active, quiescent, or remitted ulcerative colitis under IBD medications [100][101][102]. Various factors could have contributed to the differential outcome of ω-3 PUFA diet in patients such as the age, metabolic status, dose, or the impact of co-administered IBD medications. A schematic representation summarizing the antiinflammatory and antioxidative mechanisms of ω-3 PUFA through NF-κB inhibition in IEL is reported in Fig. 3. Table 1 and 2. Fig. 3 Model summarizing the immunomodulatory mechanisms of omega-3 polyunsaturated fatty acids in the intestinal epithelium. In arachidonic acid (ARA)-enriched cell membranes, during an infection or injury, pathogen-associated molecular patterns (PAMPs) or damageassociated molecular patterns (DAMPs) binds with the host-specific pattern-recognizing receptors (PRRs) and activate the nuclear factor-κB (NF-κB) signaling to release pro-inflammatory cytokines, chemokines and reactive oxygen species (ROS). Subsequently, these mediators recruit the inflammatory cells from lamina propria and exert a strong pro-inflammatory response. Pro-inflammation also damages the integrity of the epithelial barrier by disrupting the tight junction proteins. Loss of epithelial integrity aggravates inflammation by facilitating the translocation of luminal pathogens and endotoxins into the circulatory system (Black lines). Dietary supplementation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ameliorates the pro-inflammatory response by replacing ARA in specific cell membrane G-protein-coupled receptors (GPCR) and instead stimulates the production of anti-inflammatory cytokines and antioxidants (Red lines). For references, see text.

Citrus pectin Structure and molecular mechanism
CPn is a highly branched, complex heteropolysaccharide derived from the peels and pomace of citrus fruits. The structure of CPn involves a covalently linked, galacturonic acid backbone with three major polysaccharide units of homogalacturonan, rhamnogalacturonan, and substituted galacturonans [105,106]. Generally, the small intestine cannot assimilate native pectins due to its large molecular weight (60-300 kDa), complex structure, and higher degrees of esterification (DE~70%). However, pH or enzymatic treatment yields compounds of low molecular weight (15 kDa) and reduced DE (< 5%). The modified CPn exhibit enhanced intestinal absorption and transportation [107,108]. Interest in utilizing CPn as a potential feed additive is emerging from the recent pieces of evidence on its anti-inflammatory, antimicrobial, and prebiotic activities [30,34,109,110]. Owing to the complex physiochemical structure of CPn, its precise molecular mechanism is fairly established [34]. However, previous reports on its ability to interact with TLRs, Galectin-3 (Gal-3) or produce short-chain fatty acids (SCFAs) could be attributed to part of its immunomodulatory mechanisms in the intestinal barrier [30,98] [Fig. 4]. Interestingly, non-digestible carbohydrates and LPS share a structural similarity with respect to carbohydrate-containing regions that interact with IECs and other immune cells [98]. CPn belongs to the category of non-digestible carbohydrates and was shown to interact with TLR2/4 that resulted in modulating NF-κB in both IECs and immune cells [30,98,[100][101][102][105][106][107][108][109][110][111][112][113][114]. Another mechanism by which CPn controls inflammation is by inactivating Gal-3 signaling proteins. Gal-3 belongs to the lectin family, a class of carbohydrate-binding proteins that dispersed in both intracellular and extracellular regions. It possesses a unique carbohydrate-recognition domain (CRD) that binds to the β-galactoside sugars [106,115]. Gal-3 was reported to involve in the pathogenesis of microbial infections, cancer, and several other inflammatory diseases [116,117]. The mechanism by which Gal-3 regulates inflammatory response is multifaceted. In intestine, it stimulates pro-inflammatory responses by acting as ligands to TLRs of IECs [117,118] [Fig. 4B]. Previously, in rat pups, induction of experimental NEC was shown to activate Gal-3-mediated TLR4/NF-κB signaling [118]. Alternatively, Gal-3 act as PRRs to pathogens or LPS and recruit immune cells for opsonization [117] [ Fig.  4B]. Of note, orally or intravenously administered CPn are able to cross the intestinal barrier and inhibit Gal-3mediated inflammation by binding its CRD region [119,120] [Fig. 4B]. Currently, the application of CPn as Gal-3 inhibitor is emerging as a novel strategy for treating cancer and many inflammatory diseases [35,106,108,121]. From all these observations, it could be possible that certain carbohydrate regions of CPn, LPS, and Gal-3 that binds to IECs or immune cells are quite similar. This could have enabled CPn to prevent inflammation by competing with LPS or Gal-3 for the TLR-binding region [ Fig. 4A, B]. Moreover, the SCFAs produced during the fermentation of dietary fibers such as CPn was reported to improve the intestinal barrier functions [98].

Impact on intestinal barrier under normal and stress conditions
In vitro studies CPn self-assembles into dense hydrogel matrix on contact with the mucin glycoproteins of IEL [122] [Fig. 4C]. The gel-forming ability of CPn (DE94% and DE25%) was demonstrated using the commercially available mucins and on the mucosal surface of porcine colonic tissues. In this study, CPn-DE94% formed a gel matrix at the tissue surface, while CPn-DE25% permeated deeper towards the tissue wall. Besides, the net electrical charge of CPn influenced the rheological strength of the gel [123]. Following, different oligosaccharides of CPn were shown to cross the Caco-2 cell monolayer based on the degree of polymerization in a transwell culture system. Particularly, only the shortchain galectins and arabinogalactans could transverse, but not the galacturonic acid [107].
A recent study demonstrated the immunomodulatory behaviour of in vitro digested citrus pulp on the intestinal barrier using IPEC-J2 cell model. Specifically, it inhibited the gene expressions of TLR4 and CLDN-1 and instead activated NOD1 under a stress-free environment [124] (Table 3). Further, many studies demonstrated the barrier protective properties of CPn under inflammatory conditions. Accordingly, CPn with different degrees of methyl esters (DM32%, DM59%, and DM64%) secured the integrity (TEER) and reduced the permeability (lucifer yellow flux) of mouse CMT93 cell monolayer that was disrupted by the pathogenic challenge of C. rodentium. Interestingly, using a reporter cell assay, CPn was shown to activate the NF-κB/AP-1 signaling via TLR2 in the CMT93 cells independent of C. rodentium challenge. Additionally, CPn imparted anti-adhesive effects on C. rodentium by interacting with the pathogen instead of CMT93 cells [113]. Likewise, both native and enzymatically modified citrus residues prevented the Caco-2 cells from secreting IL-8 upon the pathogenic challenge of Salmonella typhimurium (S. typhimurium) and Listeria monocytogenes (L. monocytogenes) [33]. In this study, citrus treatment selectively supported the cellular adhesion of probiotics (Lacticaseibacillus casei and Bifidobacterium lactis), while repelled the pathogens (S. typhimurium and L. monocytogenes) [Fig. 4C]. Moreover, the enzymatically modified citrus residues exhibited enhanced antibacterial and prebiotic activities in comparison to the unmodified [33]. In another study, CPn (DM30%, DM56%, and DM74%) secured the integrity (TEER) of T84 cell monolayer from the barrier disrupting agent such as the phorbol esters [114].

Structure and molecular mechanism
Exosomes are membrane-bound, extracellular vesicles of 30-150 nm size and 1.13-1.19 g/mL density. It carries cargos of nucleic acids, proteins, lipids, and other signaling molecules for specific cellular functions [135]. Exosomes are either directly released from the cell membranes or produced within the cells and then externally released [136]. Most cells secrete exosomes into various body fluids including the blood and milk [136,137]. Recent bioinformatical analysis has identified the presence of numerous microRNAs (miRNAs) and peptides in the milk-derived exosomes (MDEs) relevant for developmental and immune-related activities of the intestinal barrier [38,39,138]. Generally, miRNAs are single-stranded, non-coding RNA molecules of 18-25 nucleotides long. The primary miRNA is synthesized in the nucleus and then transported to the cytoplasm, where it is processed into mature miRNA. In association with the RNA-inducing silencing complex, the mature miRNA binds to a complementary mRNA and inhibit protein synthesis [139]. Through dietary MDEs, the molecules that are involved in initiation, propagation, and resolution phases of intestinal inflammation can be controlled [ Fig.5].

Impact on intestinal barrier under normal and stress conditions
In vitro studies Dietary MDEs survives the intestinal digestion and are subsequently taken up by the IECs or transported across [140][141][142]. The miRNA sequencing of MDEs has revealed that even harsh gastric/pancreatic digestion had a limited impact on its miRNA profiles [140,141]. Further, MDE uptake is driven by receptormediated endocytosis in various cell types including the small intestinal IECs [143]. This shows how the dietary MDEs stably cross the intestinal barrier and enter systemic circulation in order to establish specific 'cell-tocell' communication. Moreover, maternal MDEs carry miRNAs, proteins, and other growth-promoting factors necessary for the maturation of infant gut immunity [144]. Several studies have demonstrated the ability of miRNAs to control the developmental and immunerelated activities of the intestinal barrier (Table 4).
Accordingly, supplementation of porcine MDEs improved the viability and proliferation of IPEC-J2 cells by increasing the gene expression of homeobox transcription factor-2 (CDX2), insulin-like growth factor-1 receptor (IGF-1R), and proliferating cell nuclear antigen (PCNA). Besides, the miRNAs targeting the p53 cell death pathway were transferred from MDEs to the IPEC-J2 cells. This subsequently downregulated the genes involved in p53 pathway such as the p53, cellsurface death receptor (FAS) and serine protease inhibitor clade E (SERPINE) [138]. Likewise, rat MDEs stimulated the viability, proliferation, and stem cell activity of rat IEC-18 cells by activating the gene expression of PCNA and leucine-rich repeat-containing G-protein coupled receptor-5 (LGR5) [145].
Interestingly, the human MDEs selectively induced proliferation and mesenchymal-like morphology in the human normal CCD841 cells over cancer LS123 cells by modulating the expression of collagen type-I protein and twist1 gene. Furthermore, tumorigenesis was suppressed only in the normal cells by downregulating the protein expression of phosphatase and tensin homolog [146]. In another human cancer cell model as LS174T, application of bovine MDEs enhanced mucin production by stimulating the expression of genes specific for goblet cell activity such as the MUC2, trefoil factor family-3 (TFF3), and glucoseregulated protein-94 (GRP94) [147]. Following, the MDEs were shown to secure the intestinal barrier from different pathogenic and non-pathogenic stress factors. Accordingly, the human pre-term MDEs highly stimulated the proliferation, migration, and healing of mechanically injured human FHC cells compared to the term-MDEs [38]. In another study, pre-treatment of bovine MDEs protected the IEC-6 cells from the oxidative damage of H 2 O 2 by enhancing cell proliferation and level of antioxidant enzymes (superperoxide dismutase, glutathione peroxidase). Additionally, the bovine MDEs inhibited the mediators of oxidative stress (ROS, malondialdehyde), LDH release, and the gene expression of NRF2 and HO-1 by altering the miRNA profiles of IEC-6 cells [153].
Further, co-administration of porcine MDEs or MDE-miRNAs with LPS decreased the impact of LPS-induced inflammation and apoptosis of IPEC-J2 cells.
Particularly, damage on cell viability, genes or proteins activated in TLR4/NF-κB pathway (TLR4, MyD88, p-IκBα, p-p65-NF-κB, p-NF-κB, NF-κB), p53 apoptotic signaling (Tp53, FAS, Caspase-3) and secretion of proinflammatory cytokines (IL-1β/-6, TNF-α) were significantly attenuated [148,149]. Similarly, in another study by the same group, co-administration of porcine MDEs with DON lowered the impact of DON-induced toxic stress in the IPEC-J2 cells. Specifically, DON-induced damage on cell viability, genes or proteins corresponding to proliferation (β-catenin, cyclin D1 [CCND1], protein kinase B [Akt]) and tight junction proteins (ZO-1, OCLN, CLDN1) were significantly recovered by the treatment of porcine MDEs. Additionally, DON-activated genes or proteins that involved in apoptosis (Tp53, p21, FAS, SERPINE1, caspase-3/-9) were suppressed by the porcine   [152] MDEs. Besides, the miRNAs targeting the p53 pathway were highly expressed in the IPEC-J2 cells postincubation with porcine MDEs [150]. In the intestinal organoids of neonatal mice, co-administration of human MDEs (from colostrum, transitional or mature milk) with LPS, markedly reduced the LPS-induced proinflammatory gene expression (TLR4, TNF-α) and morphological damage. Besides, the human MDEs stimulated the expression of markers specific for epithelial proliferation (Ki67 + cells) and regeneration (LGR5 gene) in the organoids. Amongst the three MDE-treatment groups, colostrum-MDEs exhibited enhanced cytoprotective effects [154]. Several recent studies assessed the capability of MDEs in controlling the hypoxic stress in intestinal barrier. Accordingly, pre-treatment of cow MDEs, yak MDEs or yak MDE-miRNAs enhanced the survival and proliferation (Ki67 + cells) of IEC-6 cells under hypoxic stress. Especially, the yak MDEs or MDE-miRNAs enhanced hypoxia resistance by decreasing the protein expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (VEGFA), and different apoptotic markers (p53, B-cell lymphoma 2-associated X protein [Bax], Caspase-3/-9), while upregulated prolylhydroxylases-1 (PHD-1) [39,151]. Additionally, the miRNA profiling of MDEs has disclosed the presence of miRNAs relevant for hypoxia protection and intestinal barrier development. The microscopic observations also revealed that yak MDEs were highly taken up by the cells during normoxic conditions over the cow MDEs [39,151]. Likewise, in another study, pre-treatment of human MDEs protected the human FHS-74 and rat IEC-6 cells from alternating hypoxia/reoxygenation injury by inhibiting apoptosis and enhancing the cell proliferation [152]. Similarly, both raw and pasteurized human MDEs reduced the morphologic damage, MPO activity, and the gene expression of proinflammatory IL-6, while enhanced the goblet cell abundance and MUC2 expression in the hypoxia and LPS-injured neonatal mice intestinal organoids [155].

Conclusion, limitations, and future perspectives
Based on European Food Safety Authority, the main target of an immunomodulatory feed additive is to stop the local inflammation and prevent further damage to the immune system. According to existing data, it can be concluded that the dietary omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes are able to terminate inflammation at the level of the intestinal barrier. The molecular mechanisms of these nutrients in the intestinal barrier are mainly associated with improving the expression of tight junction proteins, epithelial proliferation, enrichment of mucus layer, immunomodulation and prevention of inflammatory cell infiltration. Further, the nutrients support the maintenance of intestinal equilibrium even under stress conditions by enhancing epithelial proliferation and regeneration. Moreover, these nutrients have demonstrated considerable bioaccessibility and bioavailability across the intestinal epithelium, which is a major challenging factor in feed formulations. Although omega-3 polyunsaturated fatty acids are a well-known antiinflammatory nutrient over decades, till-date its application in controlling IBD is well established in humans, unlike animals. The lack of specific molecular studies at the intestinal level of livestock and poultry is a major setback for achieving desired health outcomes in farm animals. On the other hand, existing data on citrus pectin and milk-derived exosomes are insufficient for harnessing their application as an immunomodulatory feed additive. However, in the future, the novel property of citrus pectin to form gel matrix with mucin and selectively support the intestinal adhesion of probiotics can be utilized to reconstitute the mucosal epithelium that is damaged during infections, antibiotic therapy, or IBD. Additionally, the barrier penetrating property of low molecular weight citrus pectin can be used to target the pro-inflammatory mediators such as Galectin-3 at the local and systemic level. Intriguingly, both omega-3 polyunsaturated fatty acids and miRNA/peptide cargoes of milk-derived exosomes prevent the incidence of necrotizing enterocolitis by suppressing hypoxic stress. They can be excellent nutritional supplements especially in newborn mammals to preventing undesired inflammatory stress, hypoxia, and mortality during the pre-and post-weaning periods. In summary, all these nutrients pose a promising opportunity for controlling chronic inflammatory diseases and promote gut health in farm animals. However, it is noteworthy to mention that the molecular mechanism of these natural bioactive compounds can be diverse and poor understanding can limit its practical application. Therefore, further studies, especially using high-throughput omics technologies are necessary in order to enumerate their precise molecular mechanisms for efficient utilization in animal diets.